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Original Articles: Hepatology & Nutrition

Acetate Correction for Postabsorption Metabolism Does Not Improve the [13C]Mixed Triacylglycerol Breath Test

Slater, Christine*; Preston, Tom; Weaver, Lawrence T*

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Journal of Pediatric Gastroenterology and Nutrition: November 2006 - Volume 43 - Issue 5 - p 666-672
doi: 10.1097/01.mpg.0000232569.66699.93
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There is a well-recognized need for a noninvasive method of measuring fat digestion especially in children with cystic fibrosis (CF). Cystic fibrosis is the most common cause of exocrine pancreatic insufficiency (PI), which is expressed in children as fat maldigestion when more than 98% of pancreatic capacity for secreting enzyme is impaired. Between 10% and 15% of children with CF are pancreatic sufficient. They have milder disease expression, slower progression of lung disease, better nutritional status and higher survival rates than PI patients. The Consensus Report on Nutrition for Pediatric Patients With Cystic Fibrosis advises that pancreatic-sufficient patients should be evaluated annually for conversion to PI (1). A simple reliable test is required.

The definitive test for exocrine pancreatic function is duodenal intubation with secretin-pancreozymin stimulation (2), but this is invasive, uncomfortable, expensive and difficult to perform. The fecal elastase-1 test is the currently recommended indirect test of pancreatic function in children with CF (3). It has the advantage of requiring a single stool sample and is relatively inexpensive to assay using a commercially available kit, but does not establish the adequacy of pancreatic enzyme replacement therapy (PERT). High lipase doses (>25,000 U lipase/kg/day) have been associated with fibrosing colonopathy, which may result in intestinal obstruction secondary to colonic stricture, failure to thrive and abdominal pain (4,5). For this reason, it is recommended that lipase dose does not exceed 10,000 U/kg/day. The [13C]mixed triacylglycerol (MTG) breath test is noninvasive and safe and avoids the need for stool sampling (6,7). It has been used to assess the need for PERT in children with CF and to assess the ideal dose (8,9), but has not been widely adopted because it lacks specificity (10). A cause of this low specificity is the uncertainty as to the fate of ingested 13C.

Results of the MTG test are usually expressed as cumulative percentage dose recovered (cPDR) (6–8,11–17). Recovery of absorbed 13C in breath CO2 is less than 50% because of sequestration into organic carbon molecules via the tricarboxylic acid (TCA) cycle. Less than 5% is excreted in stool and urine in healthy subjects and in children with CF taking their normal PERT (18,19). Calculation of PDR requires knowledge of CO2 production rate (VCO2). A resting value is usually assumed (20), but this may not be appropriate, especially in children, who cannot remain at rest for prolonged periods. Inappropriate use of resting VCO2 results in an underestimation of the true PDR in breath, resulting in false-positive results (ie, apparent low recovery of 13C in breath) in some healthy subjects. The solution to this problem is either to measure VCO2 continuously during the test or to use a correction factor that avoids the need to know the actual value of VCO2.

Acetate correction factors have been used in studies of lipid and carbohydrate oxidation using intravenous infusions of 13C-labeled substrates, to account for the “missing” label (21,22). If an acetate correction factor were applied to the calculated PDR from 13C breath tests, such as the MTG test, it would not be necessary to know the actual value of VCO2. It would, however, be necessary to perform 2 tests, the second one using [1-13C]acetate. Percentage dose recovered is calculated from breath 13C enrichment multiplied by VCO2 and divided by the dose of 13C consumed (18). CO2 production rate is assumed to be the same on each occasion. If cPDR during the MTG test were divided by cPDR during the acetate test, the value of VCO2 cancels out, and therefore, it is not necessary to know its value.

The aim of this study was to explore the use of acetate correction factors with 13C breath tests and to investigate interindividual variation and postabsorptive metabolism of [13C]MTG using oral doses of [1-13C]acetate to account for the missing label.


Subjects and Study Protocol

Eight healthy adults, 9 healthy children and 3 children with exocrine PI secondary to CF agreed to take part in the study (Table 1). Adults and healthy children were recruited from among the staff at the University of Glasgow and their relatives. Children with CF were enrolled from those attending the Royal Hospital for Sick Children, Glasgow, but were free of pulmonary infection at the time of the test. The purpose of the study was carefully explained, and written informed consent was obtained from all subjects (adults and children) taking part and, where appropriate, from their parent or guardian. Subjects were free to drop out at any time. The study was carried out in accordance with the principles outlined in the Declaration of Helsinki. Ethical approval was obtained from the Yorkhill Research Ethics Committee for the study involving children and from the University of Glasgow Ethics Committee for Non Clinical Research Involving Human Subjects for the study involving adults.

Characteristics of subjects studied

13C Breath Tests

Each subject performed 2 breath tests. They were asked to avoid foods naturally enriched with 13C in the days preceding the test (23,24). After an overnight fast, subjects ingested 10 mg/kg [13C]MTG (1,3 distearyl 2-[1-13C]octanoyl-glycerol, 99 atom % 13C; Cambridge Isotopes Laboratory Inc, Andover, MA) or 1 mg/kg [1-13C]acetate (99 atom % 13C; Cambridge Isotopes Laboratory Inc), baked in a biscuit composed of oats, butter and honey (25). The dose was 13 μmol/kg body weight for both tracers. In vitro experiments had confirmed that the tracer was homogeneously distributed through the biscuit, and there was no loss during preparation (19). A drink of unsweetened orange juice or water was taken with the test meal. A light lunch, composed of foods with low 13C natural abundance (24), was taken 4 hours after the test meal. Children with CF took their usual dose of PERT with this meal, although not with the test meal. Heart rate monitors (Polar Vantage NV; Polar Electro Oy, Kempele, Finland) were worn throughout the test to estimate physical activity (26). Alveolar breath was sampled by exhalation into an Exetainer breath-sampling vial (Labco, High Wycombe, Buckinghamshire, UK) through a straw until condensation appeared on the inside wall of the vial. The cap was replaced immediately. Breath samples were collected at baseline, every 10 min for the first hour, and then every 20 min until 6 hours after the test meal during the acetate test and every 20 min for 6 hours during the MTG test. Adults remained seated as much as possible during the test. Children watched television or videos or played computer or board games. Body weight was measured (to 0.1 kg) using electronic scales (model 707; Seca Ltd, Birmingham, UK). Height was measured (to within 1 mm) using a Holtain stadiometer (Holtain, Crymych, Dyfed, UK). Body mass index (in kilograms per meter squared) was calculated in adults. In children, body mass index was expressed as SD scores relative to the UK 1990 reference data (27) using software from the Child Growth Foundation (London, UK).

Breath 13CO2 abundance was measured by continuous-flow isotope ratio mass spectrometry (CF-IRMS) (28). The CF-IRMS was composed of a 20-20 IRMS interfaced to an automated breath carbon analyzer (Europa Hydra; SerCon Ltd, Crewe, UK). Measurements were made against a reference gas traceable to international standards (29). Enrichment was calculated by subtracting the abundance of 13C in the baseline sample from that in the postdose samples (30). The percentage dose recovered (PDR) in each breath sample was calculated using the following equation:

The PDR in each sample was calculated using a resting value of VCO2 of 300 mmol CO2 h−1 m−2 body surface area (31). Body surface area was predicted using the Haycock equation (32). The cumulative excretion at 6 hours of 13C in breath CO2 (cPDR) was calculated using the trapezoidal rule. Percentage dose recovered was also calculated using nonresting VCO2 estimated from the heart rate (26).

The cPDR during the MTG test was divided by the cPDR during the acetate test and expressed as percent:


Results are expressed as mean (SD), except where stated otherwise. Differences were compared using a paired (1-sample), 2-tailed Student t test (Mintab for Windows Release 11.2). All other data manipulation was done within Excel spreadsheets (Microsoft Excel 97 SR-2).


PDR Calculated Using Resting VCO2 and Assuming the Same Value for Each Test

The cPDR in 6 hours (cPDR6h) during the [13C]MTG test was 34.8% (4.2%) in adults, 26.0% (5.7%) in healthy children and 8.9% (4.2%) in children with CF, who had PI. The maximum enrichment of 13C in breath CO2 during MTG tests ranged from 18 ppm excess 13C in a child with CF who normally took high-dose PERT to 150 ppm excess 13C in healthy adults. The precision of analysis (SD of 3 replicates of reference gas) was better than 1 ppm 13C. The cPDR6h during the [1-13C]acetate test was 32.9% (7.2%) in adults, 27.1% (5.7%) in healthy children and 24.4% (3.5%) in children with CF. The maximum enrichment of 13C in breath CO2 ranged from 72 to 220 ppm excess 13C during acetate breath tests. Children with PI caused by CF did not appear to be different from healthy children with respect to acetate metabolism (Fig. 1). The cPDR with an acetate correction was 103.1% (11.6%) in adults, 98.9% (30.3%) in healthy children and 37.7% (19.2%) in children with PI. Combining data from all healthy subjects (n = 17) gives a mean (SD) cPDR of 103.2 (25.1) and, therefore, a reference range (mean ± 2 SD) of 53% to 154%. Data for individual subjects are shown in Table 2. Regression analysis revealed a good correlation between cPDR6h MTG and cPDR6h acetate in adults (R2 = 0.58), but not in children (R2 = 0.05).

FIG. 1
FIG. 1:
Comparison of cPDR MTG and acetate. A, PDR calculated using restingVCO2 predicted from body surface area (31). B, PDR calculated using nonresting VCO2 estimated from the heart rate (26). Healthy adults (•), healthy children (□), children with CF and PI (▴), line of unity (—).
Cumulative percentage dose recovered

PDR Calculated Using Nonresting VCO2 Estimated From the Heart Rate During Each Test

When PDR was calculated using nonresting VCO2 predicted from the heart rate on each day and, therefore, not assuming a constant value either within each day or between days, the cPDR6h during the [13C]MTG test was 38.4% (5.3%) in adults, 44.2% (10.9%) in healthy children and 13.0% (6.0%) in children with CF, who had PI (Fig. 1B). The cPDR6h during the [1-13C]acetate test was 34.6% (5.6%) in adults, 45.7% (11.2%) in healthy children and 36.3% (5.1%) in children with CF. The cPDR with an acetate correction was 112.0% (13.9%) in adults, 99.2% (24.2%) in healthy children and 37.4% (19.7%) in children with PI. Combining data from all healthy subjects gives a mean (SD) cPDR of 105.2 (20.5) and, therefore, a reference range of 64% to 146%. Data for individual subjects are shown in Table 3. Correlation between cPDR6h MTG and cPDR6h acetate improved in children (R2 = 0.27), but not in adults (R2 = 0.48).

Cumulative percentage dose recovered

The ratio of cPDR using nonresting VCO2 to cPDR using resting VCO2 is an index of the physical activity level (PAL) during the test. The mean PAL for adults during both tests was 1.1, indicating that adults are able to sit quietly during the test. The mean PAL for healthy children was 1.7 and for children with CF was 1.5 during both tests, indicating (as expected) that children are not able to sit quietly for 6 hours. This would not be important, as long as the amount and pattern of activity were the same on both occasions. Indeed 5 of 8 adults and 8 of 12 children had PAL within 0.1 units on each occasion. The difference between MTG cPDR with an acetate correction calculated using resting VCO2 and using nonresting VCO2 was not significant (P = 0.57). The mean difference was 1.8% (95% confidence interval, −4.7% to 8.3%).


We have shown that the low specificity of the [13C]MTG breath test is caused by physical activity during the test, which results in dilution of labeled CO2 from the oxidation of [13C]MTG by unlabeled CO2 from the oxidation of other substrates and, hence, low 13C enrichment in breath (33). Use of resting VCO2 to calculate PDR in these circumstances is inappropriate and leads to an underestimation of the amount of tracer oxidized. Variation of physical activity between tests may also contribute to the poor reproducibility of the [13C]MTG breath test (34). In addition, differences in nutritional status on the day of the test could result in differences in intermediary metabolism.

Use of an acetate correction factor assumes that acetate and octanoate are assimilated in the same manner and at the same rate by the liver and that there are no other major routes of sequestration of label. If these assumptions were true, recovery in breath corrected for label sequestered in the body should approach 100% in healthy subjects. We have shown that the mean (SD) recovery of 13C during the [13C]MTG with an acetate correction was close to 100% in healthy subjects: 103.1% (11.6%) in adults and 98.9% (30.3%) in children, but the variance is very wide especially in children.

Quantitation of tracer excretion requires knowledge of the volume of distribution of the tracer, as well as the quantity and enrichment of the ingested dose and the enrichment in the product of oxidation. In 13C breath tests, labeled CO2 from the tracer is diluted by unlabeled CO2 from metabolism. The volume of distribution is related to VCO2. CO2 production rate is increased during physical activity and after food intake, thereby diluting breath 13CO2 enrichment. Use of resting VCO2 to calculate PDR in these circumstances will underestimate the true tracer excretion (3). In addition, recovery is never complete from 13C-labeled lipids (8,17,33–35).

Pancreatic lipases preferentially hydrolyze MTG at the sn-1 and sn-3 positions, leaving 2-[1-13C]octanoyl-glycerol. Hydrolysis of medium-chain 2-monoacylglycerol is rapid and complete (36), releasing [1-13C]octanoate, which is absorbed and reaches the liver via the portal vein. In the mitochondria, the 13C-label is removed by β-oxidation resulting in [1-13C]acetyl–coenzyme A (CoA), which can enter the TCA cycle. Some 13C leaves the TCA cycle as 13CO2, which equilibrates with the bicarbonate pool before being excreted in breath as labeled CO2 or urine as labeled bicarbonate, or it can enter the urea cycle and be excreted in urine as labeled urea. Losses in urine account for less than 5% of ingested 13C (19). The remainder is retained in the body during the timescale of a typical breath test (∼6 hours). The presence of concurrent liver disease does not affect the recovery of 13C during the MTG test in children with CF (15).

Use of an acetate correction factor to account for tracer retained in the body assumes that CO2 production rate is the same during both tests. Figure 1 shows cPDR during the [13C]MTG test plotted against cPDR during the [13C]acetate test. When data are plotted in this manner, that from pancreatic insufficient subjects should form a distinct cluster away from that of healthy subjects, if the assumptions discussed above are correct, and the gradient of the regression line through data from healthy subjects should be unity with zero intercept. In Figure 1A, PDR is calculated using a predicted value of resting VCO2. In Figure 1B PDR is calculated using nonresting VCO2 predicted from the heart rate (26). The discrimination of the test improved when nonresting VCO2 was used in the calculation of PDR (Fig. 1B), suggesting that physical activity and, therefore, VCO2 were not the same on each occasion. However, the broad range of the recovery of 13C in the breath of healthy subjects after applying an acetate recovery factor, even when physical activity is taken into account, suggests that this is not the only factor influencing the recovery of 13C in breath.

An assumption in the use of an acetate correction factor is that MTG, octanoic acid and acetate are all metabolized in the same manner and at the same rate by the liver. However, this assumption may not be valid when the tracer is taken orally. We have observed lower recovery of deuterium (2H) in body water after ingestion of (2H3)acetate, than free (2H15)octanoate or esterified (2H15)octanoate in the form of (2H15)MTG (37). From studies of body composition and total energy expenditure by isotope dilution, using 2H2O, it is known that 2H sequestration into organic molecules is low (∼4 %) (38,39) and is not influenced by factors that affect VCO2 such as food intake or physical activity.

Differential metabolism could occur in the liver because of compartmentation of enzymes within hepatocytes. Fatty acids must be activated before metabolism. Acyl-CoA synthetases catalyze the reaction of the fatty acids with CoA. There are several acyl-CoA synthetases with affinities for fatty acids of different chain length located in distinct parts of the cell. In the liver, octanoic acid is activated mainly in the mitochondria of the hepatocyte (40), which is also the site of β-oxidation and the TCA cycle. Acetyl-CoA synthetase is present in the cytosol of hepatocytes, but not in the mitochondria (41). In the liver, acetyl-CoA produced in the cytosol cannot cross the mitochondrial membrane, and therefore, exogenous acetate cannot be oxidized in the liver, but can be transported to other extrahepatic tissues, for example, skeletal muscle for activation and oxidation (42). Under conditions of high rates of fatty acid oxidation, the production of endogenous acetyl-CoA can exceed the capacity of the TCA cycle, and the excess is exported from the hepatocyte as acetylcarnitine for metabolism in other tissues (41).

Exogenous acetate can be activated in the cytosol of hepatocytes and be incorporated into long-chain fatty acids by de novo lipogenesis (42–44) and, hence, triacylglycerol, phospholipids and very low-density lipoprotein, or it can be exported from the liver and oxidized in other tissues, for example, skeletal muscle. In contrast, octanoate is mainly oxidized in the liver, although incorporation into long-chain fatty acids by elongation is also possible (45,46). The balance between lipogenesis and oxidation of acetate at any one time depends on the physical activity and nutritional status of the individual. Low recovery of 13C from acetate compared with MTG results in an overestimation after the acetate correction has been applied and occurs under conditions of lipogenesis. High recovery of acetate compared with MTG could occur under nonresting conditions when exogenous acetate is oxidized in skeletal muscle, while endogenous acetate (from octanoate) is sequestered via TCA cycle intermediates into anabolic pathways.

A theoretical alternative might be to use a C1-labeled, medium-chain fatty acid (butyrate, hexanoate or octanoate) that is subject to β-oxidation, to correct for label retained in the body, but there would be severe palatability problems with this approach, when the tracer is given orally. In addition, variation in physical activity between tests, especially in children, and the need to perform 2 tests make this approach impractical. Acetate correction factors offer no advantage over the use of heart rate monitors to estimate PAL during 13C breath tests (26,33). Simultaneous use of [14C]MTG with [1-13C]octanoic acid could be a practical approach in adults, although not in children, as one of the advantages of 13C breath tests is the avoidance of radiation hazards.

In accounting for the missing label, we have shown that differences in PAL during 13C breath tests are more important than differences in intermediary metabolism (Fig. 1). The wide variance in cPDR can be accounted for by differences in intermediary metabolism, but improvements in specificity are due to use of nonresting VCO2 to calculate PDR. Use of 2H-labeled compounds, in which the label is distributed in body water and recovery is not influenced by physical activity, could prove useful in studies of macronutrient oxidation (47).


The need to perform 2 tests, variation in physical activity between tests and differences in intermediary metabolism preclude the use of acetate correction factors when using [13C]MTG to assess intraluminal fat digestion, especially in children.


The authors received financial support from the MRC Joint Research Equipment Initiative and the University of Glasgow and thank Cambridge Isotopes Laboratory Inc, Andover, MA, for donating the [13C]MTG used in this study.


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[1-13C]acetate; 13C breath test; MTG breath test; Acetate correction factor; Fat digestion; Intermediary metabolism; Acetate metabolism

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